Hydrogen course - Reykjavík 2010
Biological hydrogen production
• Hydrogen as an energy carrier
• Routes for hydrogen production
• Fermentation – Mesophilic vs.
Thermophilic • Examples of data
Hydrogen course - Reykjavík 2010
Hydrogen as energy carrier
• Hydrogen – Clean renewable energy source – Highest energy density of any fuel (carbon
free) – Contributes substantially to the reduction of
greenhouse gas emissions. – H2 from renewable sources might be
considered as the ultimate clean and climate neutral energy system
Hydrogen course - Reykjavík 2010
Comparison of energy and emissions of combustable fuels
Fuel type Energy/unit (MJ/kg) Energy/vol (MJ/l) Kg of C release/kg fuel used
Hydrogen gas 120 2 0 Hydrogen liquid 120 8,5 0
Coal 15-30 0.6 Natural gas 33-50 9 0.46 Petrol 40-43 31,5 0.86 Oil 42-45 38 0.84 Diesel 43 35 0.9 Bio-Diesel 37 33 0.5 Ethanol 21 23 0.5
Charcoal 30 0.5
Agric. Residues 10-17 0.5 Wood 15 0.5 From Vijayaraghavan & Soom, 2005 (Int. J. Hydrogen Energy)
Hydrogen course - Reykjavík 2010
Unit cost of energy obtained by different processes
Type of Energy Conversion effciency (%) Unit cost (US$) H2 (photobiological) 10 10 H2 (fermentation) 10 40
H2 (coal/biomass) 4
H2 (electrolysis) 10 H2 (thermal decompostion 13
H2 (photochemical) 21 Ethanol (fermentation) 15-30 32 Gasoline 6 From: Das & Veziroglu, 2001 (Int. J. Hydrogen Energy)
Hydrogen course - Reykjavík 2010
Biological hydrogen production - routes
• Hydrogen production by – Direct biophotolysis – Indirect biophotolysis – Photo-fermentation – Dark-fermentation
Hydrogen course - Reykjavík 2010
Biological hydrogen production - routes
Hydrogen course - Reykjavík 2010
Dark fermentation • Hydrogen can be produced by anaerobic
bacteria, grown in the dark on carbohydrate-rich substrates – Mesophilic (25–40°C) – Thermophilic (40–65°C) – Extreme thermophilic (65–80°C) – Hyperthermophilic (>80°C)
Hydrogen course - Reykjavík 2010
Anaerobic digestion
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Glycolysis
Fermentation
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Carbohydrates
Pyruvate
Ethanol Lactate Butyrate Acetate
Hydrogen - Acetate route
Reduced end products
Thermophiles
Mesophiles
Extremophiles
Hydrogen course - Reykjavík 2010
Hydrogen production from carbohydrates
• Carbohydrates yield different amounts of H2 depending on the fermentation pathway and end products – C6H12O6 + 2 H2O 2 CH3COOH + 2 CO2 + 4 H2 – C6H12O6 + 4 H2O CH3CH2CH2COOH + 2CO2 + 2 H2
• Acetate highest hydrogen yields • In practice – mixture of Ac/But and other
products lower yields
Hydrogen course - Reykjavík 2010
Hydrogen production from carbohydrates
• Processes important for H2 production – pH – HRT (in continuous cultures) – pH2
– Initial substrate concentrations – Fermentation end products depend on the
environmental conditions
Hydrogen course - Reykjavík 2010
Hydrogen production from carbohydrates
• Reduced fermentation end products are produced when H2 accumulates – Etanol, Lactate, Butanol, Alanine
• To maximize the yield of H2 the metabolism must be directed away from alcohols and reduced acids
Hydrogen course - Reykjavík 2010
Fermentation
• The majority of microbial H2 production is driven by the anaerobic metabolism of pyruvate formed during the catabolism of various substrates.
• The breakdown of pyruvate is catalyzed by one of the two enzyme systems: – Pyruvate: formate lyase (PFL)
• (Pyruvate + CoA) acetyl-CoA + formate – Pyruvate: ferredoxin (avodoxin oxido
reductase (PFOR) • Pyruvate + CoA + 2 Fd (ox) acetyl-CoA +
CO2 + 2 Fd (red)
Hydrogen course - Reykjavík 2010
Fermentation • Pyruvate acetyl-CoA ATP + formate/reduced ferredoxin H2 • The overall yields are relatively low,
– 1-2 H2 per molecule of pyruvate. • fermentations have been optimized by evolution to produce
biomass and not H2. • Thus, a portion of the substrate (pyruvate) is used in both cases to
produce ATP, giving a product (acetate) that is excreted.
Hydrogen course - Reykjavík 2010
Fermentation - thermodynamics
• The major issue is the feasibility of a dark fermentative reaction yielding close to the 12 mol H2 stored in each molecule of glucose metabolized.
• From a thermodynamic perspective, the most favourable products from the breakdown of 1 mol of glucose gives rises to 2 mol of acetate and 4 mol of H2. – In reality: Maximum 3.3 mol H2
Hydrogen course - Reykjavík 2010
pH2
• pH2 is extremely important for H2 production • H2 synthesis pathways are sensitive to H2 conc.
and are subject to end product inhibition • Previously: ↑ H2 ↑ pH2 slower down H2
production • Thermodynamics: ↑T°C pH2 has less effect • Thus at higher temperatures oxidative reactions
can occur to a more extent before reduced compound are produced AND inhibition starts at higher pH2
Hydrogen course - Reykjavík 2010
Mesophilic hydrogen producing bacteria
• Strict anaerobes – Clostridium – Citrobacter – Klebsiella – Enterobacter
Thermophilic hydrogen producing bacteria
• Clostridium • Caloramator • Thermoanaerobacter • Thermoanaerobacterium • Caldicellulosiruptor • Thermotoga
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Results
• Samples were collected in four trips from three different geothermal areas in 2004, 2005, 2007 and 2009
Results
• Isolations of hydrogen producing strains • Basic physiological characteristics • Substrate spectrum • Influence of initial substrate concentrations • Influence of pH2
• Continuous culture • Complex biomass studies
Hydrogen course - Reykjavík 2010
Physiological data of sampling sites
• Örlygsson and Baldursson, 2007.
• - 4 strains isolated • AK1 and AK14 (both
Clostridium) – Isolated from Grensdalur
(Hveragerdi)
• AK15 (Clostridium) and AK17 (Thermoanerobacterium) – Isolated from Hell (Víti)
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Sampling and environment
Hydrogen course - Reykjavík 2010
Light microscope
Electron microscopy
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Physiological data
AK1 AK14 AK15 AK17
Topt 45 45-50 55 58
Tmax 55 55.68 80 72
pHopt 7.0 - 8.0 7.0 7.0 4.5-6.5
µmax 0.16 0.44 0.25 0.4
Gen time 4.0 1.6 2.7 2.0
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Hydrogen production rates
0.96 mmol/L 4.34 mmol/L
0.56 mmol/L 1.91 mmol/L
Hydrogen course - Reykjavík 2010
Fermentation end products
AK14
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Continuous culture
• Ethanol and Hydrogen Production by Two Thermophilic, Anaerobic Bacteria Isolated From Icelandic Geothermal Areas – Biotechnology and Bioengineering
• Two bacterial strains were isolated from two sediment samples collected in the Krafla area (Víti=Hell) in NE-Iceland
– AK15: 60°C; pH = 8.6 – AK17: 70°C; pH = 6.5
Hydrogen course - Reykjavík 2010
Batch fermentation patterns from glucose and xylose by the isolate AK17
• Glucose – – Hydrogen main fermentation product
• 0.4-1.2 mol H2/mol glucose – Ethanol
• 1.2-1.6 mol EtOH/mol glucose – Acetate
• 0.5 mol/mol glucose • Xylose
– Hydrogen • 0.9-1.0 mol H2/mol xylose .
– Acetate was the main soluble metabolite – Ethanol
• 1.0-1.1mol/mol xylose – Acetate - less
Hydrogen course - Reykjavík 2010
EtOH 30 mM
Acetate 10 mM
Compared to literature
Hydrogen course - Reykjavík 2010
Bioprospecting hydrogen producers
• Icelandic Agricultural Sciences, 2010 • Purpose – Isolate and characterize
thermophilic saccharolytic bacteria • New enrichment experiments
– Hveragerdi (SW Iceland) and Krafla (NE Iceland)
Hydrogen course - Reykjavík 2010
Methods... • Geysers at 50 – 80°C and pH 3 – 7. • Carbon sources: glucose, xylose, cellulose, pectin, xylan • # samples = 47 x 6 282 • Glucose and xylose = 100 mM • Polymers = 5 g/L • Growth follwed by H2 measurements • Best growth end point dilutions and agar plates • phylogenetic analysis (16S rRNA)
Hydrogen course - Reykjavík 2010
Selection of strains
• Aim: Characterize all strains concerning end product formation
• The best strains (↑EtOH, ↑H2) chosen • Plus = growing on many types of sugars and
polysaccharides • Hydrogen producers: EtOH/Acetate = < 1 • Ethanol producers: EtOH/Acetate = > 3
Hydrogen course - Reykjavík 2010
Enrichment cultures
Hydrogen course - Reykjavík 2010
Results • Low temperature hot springs
– Thermoanaerobacterium, Clostridium, Paenibacillus, Caloramator
– Sugars ethanol, butyrate (acetate, H2)
• High temperature hot springs – Thermoanerobacter ,
Caldicellulosiruptor, – Sugars acetate + H2
• A clear correlation between phylogeny (types of bacteria), temperature and end product formation
• Culture collection obtained Hydrogen course - Reykjavík
2010
Example of AK14
Hydrogen course - Reykjavík 2010
• A moderate thermophilic bacterium – belongs to Clostridium
• Icelandic Agricultural Sciences 2010 • Acetate/butyrate fermentation spectrum
– End product formation from glucose and xylose (both 20 mM)
Substrate spectrum from 20 mM or 3 g/L of various carbohydrates
Hydrogen course - Reykjavík 2010
0
10
20
30
40
50
60
Xylose Glucose Fructose Galactose Mannose Sucrose Starch Xylan
End
prod
ucts
(mM
or m
mol
/L)
Ethanol (mM)
Acetate (mM)
Butyrate (mM)
H2 (mmol/L)
Initial glucose concentrations
Hydrogen course - Reykjavík 2010
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
0 5 10 30 50 100 200 400
End
prod
ucts
(mm
ol/L
)
Glucose (mM)
Ethanol
Acetate
Butyrate
Hydrogen
pH2
• By simply using different gas-to-liquid ratios get insight into the effects of hydrogen in the gas phase on end product formation and the inhibition of hydrogen production
• Use 20 mM Glucose • L-G ratios: 3.0 0.02
– Example: 3.0 = 90 mL liquid and 30 mL gas
Hydrogen course - Reykjavík 2010
pH2
Hydrogen course - Reykjavík 2010
1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9
0 2 4 6 8
10 12 14 16
0 1 2 3 4
Hyd
roge
n (m
ol/m
ol g
ucos
e)
End
prod
ucts
(mM
)
Liquid/Gas phase ratio
Ethanol (mM) Acetate (mM)
Butyrate ( mM) Hydrogen (mmol)
pH2
• As expected, lower H2 yields followed the decrease in acetate and butyrate formation as against an increase in ethanol production.
• Using the fermentation data from the lowest and highest L/G ratios the following equations are observed:
Hydrogen course - Reykjavík 2010
But....
• .... H2 production from monosugars is one thing next step is to go to complex biomass Production of second generation of hydrogen
Hydrogen course - Reykjavík 2010
2nd generation – a closer look • Production of ethanol from waste
material • Lignocellulose
– Cellulose, hemicellulose and lignin – Grass, starw, saw, etc – 2° - generation hydrogen
production • Is it possible to produce hydrogen from
such biomass? • The basic structure of lignocellulose is
the same, i.e. polymetric sugars that can be converted to monosugars and ferment to ethanol.
• More expensive, more pretreatment, and enzymes.
Hydrogen course - Reykjavík 2010
Hydrogen course - Reykjavík 2010
Experimental
Hydrogen course - Reykjavík 2010
Experimental
Preparation of HL • Hydrolysates (HL) were made from different biomasses:
– Whatman filter paper (cellulose), – hemp (Cannabis Sativa L.) – leaves and stem fibres – newspaper with ink (NPi), – barley straw (BS) (Hordeum vulgare L.) and – grass (Phleum pratense L.).
• Chemical pretreatment acid and base • Heat autoclaving for 30 minutes (121°C). • Enzymes (Celluclast® and Novozyme 188) • Lignocellulosic hydrolysates ready for fermentation
Hydrogen course - Reykjavík 2010
Second generation of H2
Hydrogen course - Reykjavík 2010
0
5
10
15
20
25
30
35
Ethanol (mM)
Acetate (mM)
Butyrate (mM)
Hydrogen (mmol/L)
Second generation of H2
• The stochiometry for pure glucose and the cellulose hydrolysate (HL) experiments are:
• The end product formation in the cellulose hydrolysate experiment was slightly higher except for ethanol and carbon recovery was 80%.
• The hydrogen yield on cellulose hydrolysate was 1.39 mol-H2/mol-glucose equivalent
Hydrogen course - Reykjavík 2010
Future aspects - questions
• Initial substrate concentratins for thermophiles seem to be a problem fed batch or continuous
• Lignocellulose – use waste first ! • Hydrogen removal or use bacteria that tolerate
high partial pressures of hydrogen
Hydrogen course - Reykjavík 2010
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